Zinnia line SAKZIN020

ABSTRACT

A Zinnia plant designated SAKZIN020 is disclosed. Embodiments include seeds of Zinnia SAKZIN020, plants of Zinnia SAKZIN020, to plant parts of Zinnia SAKZIN020, and methods for producing a Zinnia plant produced by crossing Zinnia SAKZIN020 with itself or with another Zinnia line. Embodiments include methods for producing a Zinnia plant containing in its genetic material one or more genes or transgenes and transgenic Zinnia plants and plant parts produced by those methods. Embodiments also relate to Zinnia lines, breeding varieties, plant parts, and cells derived from Zinnia SAKZIN020, methods for producing other Zinnia lines or plant parts derived from Zinnia SAKZIN020, and the Zinnia plants, varieties, and their parts derived from use of those methods. Embodiments further include hybrid Zinnia seeds, plants, and plant parts produced by crossing Zinnia SAKZIN020 with another Zinnia line.

BACKGROUND

All publications cited in this application are herein incorporated byreference. Zinnia is a species of flowering plants in the familyCompositae (Asteraceae).

Zinnia s can be propagated from seed, cuttings, and tissue culture.Seed, cuttings and tissue culture germination protocols for Zinnia arewell-known in the art.

Zinnia is an important and valuable ornamental plant. Thus, a continuinggoal of ornamental plant breeders is to develop plants with novelcharacteristics, such as color, growth habit, and hardiness. Toaccomplish this goal, the breeder must select and develop plants thathave traits that result in superior Zinnia varieties.

The foregoing examples of the related art and limitations relatedtherewith are intended to be illustrative and not exclusive. Otherlimitations of the related art will become apparent to those of skill inthe art upon a reading of the specification.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file may contain one or more drawings executedin color and/or one or more photographs. Copies of this patent or patentapplication publication with color drawing(s) and/or photograph(s) willbe provided by the Patent Office upon request and payment of thenecessary fee.

FIG. 1 shows the overall plant habit of the plant grown in a pot.

FIG. 2 shows a close-up of a mature flower of the plant.

SUMMARY

The following embodiments and aspects thereof are described inconjunction with systems, tools and methods which are meant to beexemplary, not limiting in scope. In various embodiments, one or more ofthe above-described problems have been reduced or eliminated, whileother embodiments are directed to other improvements.

According to one embodiment, there is provided a Zinnia plant which isvalued as breeding line enabling the development of superior ornamentalZinnia plants.

Another embodiment discloses a Zinnia plant and seed, wherein a sampleof representative seed of said Zinnia is deposited with a BudapestDepository.

Another embodiment relates to tissue culture produced from protoplastsor cells from the Zinnia plants disclosed in the subject application,wherein said cells or protoplasts are produced from a plant partselected from the group consisting of pollen, ovules, embryos,protoplasts, meristematic cells, callus, pollen, leaves, ovules,anthers, cotyledons, hypocotyl, pistils, roots, root tips, flowers,seeds, petiole, seed, and stems.

Another embodiment relates to a plant, or a part thereof, produced bygrowing Zinnia SAKZIN020, wherein the plant part comprises at least onecell of Zinnia SAKZIN020.

Another embodiment relates to a tissue or cell culture of regenerablecells produced from the plant of SAKZIN020 and a Zinnia plantregenerated from the tissue or cell culture of SAKZIN020.

Another embodiment relates to a method of asexually/vegetativelypropagating the plant of SAKZIN020, comprising the steps of: collectingtissue or cells capable of being propagated from a plant of SAKZIN020;cultivating said tissue or cells to obtain proliferated shoots; androoting said proliferated shoots to obtain rooted plantlets; orcultivating said tissue or cells to obtain proliferated shoots, or toobtain plantlets and a plant produced by growing the plantlets orproliferated shoots of said plant.

A further embodiment relates to a method for producing a Zinnia seed orembryo, wherein the method comprises crossing a SAKZIN020 plant with adifferent Zinnia plant and harvesting the resultant Zinnia seed embryo.

A further embodiment relates to a method for developing a Zinnia plantin a Zinnia plant breeding program, comprising applying plant breedingtechniques comprising crossing, recurrent selection, mutation breeding,wherein said mutation breeding selects for a mutation that isspontaneous or artificially induced, backcrossing, pedigree breeding,marker enhanced selection, haploid/double haploid production, ortransformation to the Zinnia plant of SAKZIN020, or its parts, whereinapplication of said techniques results in development of a Zinnia plant.

A further embodiment relates to a method of introducing a mutation intothe genome of a Zinnia plant SAKZIN020, said method comprisingmutagenesis of the plant, or plant part thereof, of SAKZIN020, whereinsaid mutation mutagenesis is selected from the group consisting oftemperature, long-term seed storage, tissue culture conditions, ionizingradiation, chemical mutagens, and targeting induced local lesions ingenomes, and wherein the resulting plant comprises at least one genomemutation and producing plants therefrom.

A further embodiment relates to a method of editing the genome of Zinniaplant SAKZIN020, wherein said method is selected from the groupcomprising zinc finger nucleases, transcription activator-like effectornucleases (TALENs), engineered homing endonucleases/meganucleases, andthe clustered regularly interspaced short palindromic repeat(CRISPR)-associated protein9 (Cas9) system, and plants producedtherefrom.

A further embodiment relates to a Zinnia seed produced by growingSAKZIN020.

A further embodiment relates to a method of producing a Zinnia plant, orpart thereof, by growing a seed produced on SAKZIN020.

In addition to the exemplary aspects and embodiments described above,further aspects and embodiments will become apparent by study of thefollowing descriptions.

DETAILED DESCRIPTION

Zinnia variety SAKZIN020 disclosed in the present application has shownuniformity and stability, as described in the following section viavegetative cuttings and tissue culture. Zinnia variety SAKZIN020disclosed in the present application has been asexually reproduced asufficient number of generations with careful attention to uniformity ofplant type and has been increased with continued observation foruniformity. Additionally, Zinnia variety SAKZIN020 produces viablepollen and is capable of being used as a parental line in breedingprograms.

Zinnia L. (genus Zinnia) is any made up of about 22 species of herbs andshrubs constituting the genus Zinnia of the family Asteraceae(Compositae) and native primarily to North America. They are perennialwhere they are native, from the southern United States to Chile, beingespecially abundant in Mexico, but are annual elsewhere. Zinnias havestiff, hairy stems and oval or lance-shaped leaves arranged oppositeeach other and often clasping the stem. The numerous garden varietiesgrown for their showy flowers are derived from the species Zinniaviolacea (Z. elegans). The solitary flower heads are borne at the endsof branches, growing at the junction.

The common zinnia, [formerly Z. elegans Jacq., currently Zinnia violacea(McVaugh, 1984),] is subject to attack by three major pathogens:Alternaria zinniae (Alternaria Alternaria blight), Erysiphecichoracearum (powdery mildew), and Xanthomonas campestris pv. zinniae(bacterial leaf and flower spot) (Andersen, 1971; Jones and Strider,1979; Lipschutz, 1965; Torres, 1963). These pathogens incite moderate tosevere epiphytotics within Z. violacea plantings, resulting in plantlosses, decreased ornamental value, or both. The narrow-leaved zinnia,[Z. angustifolia (McVaugh, 1984)] is highly resistant to these pathogens(Andersen, 1971; Jones and Strider, 1979; Lipschutz, 1965; Torres, 1963)and is a promising source of resistance genes for Zinnia breedingprograms. Crosses between Z. angustifolia (2n=2x=22) and Z. violacea(2n=2x=24) yield infertile allodiploids (2n=2x=23) (Boyle and Stimart,1982; Terry-Lewandowski et al., 1984). Partially fertile, true-breedingallotetraploids (2n=4x=46) have been produced by treating allodiploidswith colchicine (Boyle and Stimart, 1982; Terry-Lewandowski et al.,1984). Allotetraploids of Z. angustifolia and Z. violacea have beengiven the collective name Z. marylandica (Spooner et al., 1991).Terry-Lewandowski and Stimart (1983) found that Z. marylandica plantsdisplayed high levels of resistance to A. zinniae and E. cichoracearum,and moderate to high levels of resistance to X. campestris pv. zinniae.

It is reported that the number of numbers of chromosomes in Zinniaspecies are reported as follows: Z. angustifolia, 2n=22, Z. haageana,2n=24, Z. violacea (elegans), 2n=24 and the interspecific hybrid (Z.violacea×Z. angustifolia), namely Z. marylandica 2n=4x=46, therefore theestimated numbers of chromosome of the PROFUSION series Zinnia should be2n=4x=46.

Boyle (1996) obtained BC1 hybrids when Z. marylandica was crossed withautotetraploid forms of Z. angustifolia (2n=4x=44) and Z. violacea(2n=4x=48), hereinafter referred as BC1 hybrid. Thomas H. Boylesuggested that one Z. angustifolia genome in BC1 allotetraploids issufficient to confer resistance to A. zinniae and E. cichoracearum, butat least two Z. angustifolia genomes are required in BC1 allotetraploidsto provide resistance to X. campestris pv. zinniae. and determined thereactions of BC1 plants to A. zinniae, E. cichoracearum, and X.campestris pv. zinnia.

Data in Table 1 were taken in July 2018 in Salinas, Calif. Colorreferences are to the Royal Horticultural Society Colour Chart, 4thedition (2001). Anatomic labels are from The Cambridge IllustratedGlossary of Botanical Terms, by M. Hickey and C. King, CambridgeUniversity Press.

TABLE 1 VARIETY DESCRIPTION INFORMATION Classification: Family:Astcraccac Species: Zinnia interspecific hybrid (Z. elegans × Z.angustifolia) Plant: Species: Zinnia hybrida (Z. elegans × Z.angustifolia) Flower type: Crested, scabiosa (Wind Witch) Days fromemergence to first flower: 60 Season: Long, continuous flowering Numberof primary branches: 6 Number of secondary branches: 5 Number oftertiary branches: 12 Days from emergence of first flower: 60 Season:Long, continuous flowering Number of internodes on main stalk: 7 Lengthof internodes between 1st and 2nd nodes: 5.0 mm Diameter between 1st and2nd nodes: 8.0 mm Habit: Compact Width: 28.0 cm Height: 23.5 cmPubescence: Pubescent Stem color: RHS 114B (Yellow-Green) Leaf: Shape:Lanceolate Width: 2.5 cm Length: 6.2 cm Dorsal surface pubescence:Pubescent Ventral surface pubescence: Pubescent Color: Upper: RHS 137B(Green) Lower: RHS 147B (Yellow-Green) Base: RHS 187A (Greyed-Purple)Flowers: Length of cut flower (from head to first branch): 11.0 cmAverage number of flowers per plant: 22 Diameter of Head: 6.3 cm StemRigidity: Flexible Stem Brittleness: Brittle Type: Semi-single (manyrows of rays) Bud: Length: 1.3 cm Width: 1.4 cm Color: RHS 144B(Yellow-Green) with RHS N186A (Greyed-Purple) Ray floret: Shape: FlatDorsal surface pubescence: Glabrous Ventral surface pubescence:Pubescent Dorsal surface luster: Dull Ventral surface luster: Dull Apex:Obtuse Margin: Notched Color:  Apex half, dorsal side: RHS 14A  Apexhalf, ventral side: RHS 145B  Base half, dorsal side: RHS 60A  Basehalf, ventral side: RHS 155C  Background, ventral side: RHS 59B  Spots,ventral side: RHS 59B Disk Florets: Presence: Present, conspicuous Type:Quilled Color: Closest to RHS N172 (Greyed-Orange) with RHS 21A(Yellow-Orange) and RHS 187A (Greyed-Purple) at the center Seed: Presentand produced

SAKZIN020 is most similar to the commercial unpatented Zinnia varietyPAS951097′, commercially known as ‘Zahara Sunburst’; however, there aredifferences in the flower color as described in Table 3 (colorreferences are to the Royal Horticultural Society Colour Chart, 4^(th)edition):

TABLE 2 Comparison with commercial line Characteristic ‘SAKZIN020’‘Zahara Sunburst’ Disc Floret Closest to RHS N172 Closest to N25C withcolor With RHS 21A and N163C at center RHS 187A at center Ray ColorRHS14A (Yellow-Orange) RHS N25D (Orange) with with RHS 60A (Red-Purple)RHS N34A (Orange-Red) Flower Size 6.3 cm 7.2 cm

Further Embodiments

Breeding with Zinnia SAKZIN020

The goal of ornamental plant breeding is to develop new, unique andsuperior ornamental plants. The breeder initially selects and crossestwo or more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selection, selfing and mutations. Therefore, a breeder will neverdevelop the same genetic variety, having the same traits from the exactsame parents.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions and further selections arethen made during and at the end of the growing season. The varietiesthat are developed are unpredictable because the breeder's selectionoccurs in unique environments with no control at the DNA level, and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same variety twice by using the sameoriginal parents and the same selection techniques. Thisunpredictability results in the expenditure of large amounts of researchmonies to develop superior Zinnia lines.

Breeding programs combine desirable traits from two or more varieties orvarious broad-based sources into breeding pools from which varieties aredeveloped by selfing and selection of desired phenotypes. Pedigreebreeding is used commonly for the improvement of self-pollinatingplants. Two parents that possess favorable, complementary traits arecrossed to produce an F₁. An F₂ population is produced by selfing one orseveral F_(1s). Selection of the best individuals may begin in the F₂population; then, beginning in the F₃, the best individuals in the bestfamilies are selected. Replicated testing of families can begin in theF₄ generation to improve the effectiveness of selection for traits withlow heritability. At an advanced stage of inbreeding (i.e., F₆ and F₇),the best lines or mixtures of phenotypically similar lines are testedfor potential release as new varieties.

Using Zinnia SAKZIN020 to Develop Other Plants

SAKZIN020 plants can also provide a source of breeding material that maybe used to develop new Zinnia plants and varieties. Plant breedingtechniques known in the art and used in a Zinnia plant breeding programinclude, but are not limited to, recurrent selection, mass selection,bulk selection, hybridization, mass selection, backcrossing, pedigreebreeding, open-pollination breeding, restriction fragment lengthpolymorphism enhanced selection, genetic marker enhanced selection,making double haploids, mutagenesis and transformation. Oftencombinations of these techniques are used. The development of Zinnialines in a plant breeding program requires, in general, the developmentand evaluation of homozygous varieties. There are many analyticalmethods available to evaluate a new variety. The oldest and mosttraditional method of analysis is the observation of phenotypic traits,but genotypic analysis may also be used.

Additional Breeding Methods

Any plants produced using the SAKZIN020 plants disclosed in the presentapplication as at least one parent are also an embodiment. These methodsare well-known in the art and some of the more commonly used breedingmethods are described herein. Descriptions of breeding methods can befound in one of several reference books (e.g., Allard, “Principles ofPlant Breeding” (1999); and Vainstein, “Breeding for Ornamentals:Classical and Molecular Approaches,” Kluwer Academic Publishers (2002);Callaway, “Breeding Ornamental Plants,” Timber Press (2000).

Breeding steps that may be used in the Zinnia SAKZIN020 plant breedingprogram can include for example, pedigree breeding, backcrossing,mutation breeding, and recurrent selection. In conjunction with thesesteps, techniques such as RFLP-enhanced selection, genetic markerenhanced selection (for example, SSR markers), Gene Editing and themaking of double haploids may be utilized.

As used herein, the term “plant” includes plant cells, plantprotoplasts, plant cell tissue cultures from which SAKZIN020 plants canbe regenerated, plant calli, plant clumps, and plant cells that areintact in plants or parts of plants, such as pollen, ovules, embryos,protoplasts, meristematic cells, callus, pollen, leaves, ovules,anthers, cotyledons, hypocotyl, pistils, roots, root tips, seeds,flowers, petiole, shoot, or stems and the like.

Pedigree Breeding

Pedigree breeding starts with the crossing of two genotypes, such asZinnia SAKZIN020 and another different Zinnia plant having one or moredesirable characteristics that is lacking or which complements theZinnia SAKZIN020 phenotype. If the two original parents do not provideall the desired characteristics, other sources can be included in thebreeding population. In the pedigree method, superior plants are selfedand selected in successive filial generations. In the succeeding filialgenerations, the heterozygous condition gives way to homogeneousvarieties as a result of self-pollination and selection. Typically, inthe pedigree method of breeding, five or more successive filialgenerations of selfing and selection is practiced: F₁ to F₂; F₂ to F₃;F₃ to F₄; F₄ to F₅; etc. After a sufficient amount of inbreeding,successive filial generations will serve to increase seed of thedeveloped variety. Preferably, the developed variety compriseshomozygous alleles at about 95% or more of its loci.

Backcross Breeding

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous variety orinbred line which is the recurrent parent. The source of the trait to betransferred is called the donor parent. After the initial cross,individuals possessing the phenotype of the donor parent are selectedand repeatedly crossed (backcrossed) to the recurrent parent. Theresulting plant is expected to have the attributes of the recurrentparent and the desirable trait transferred from the donor parent. Thisis also known as single gene conversion and/or backcross conversion.

In addition to being used to create a backcross conversion, backcrossingcan also be used in combination with pedigree breeding. As discussedpreviously, backcrossing can be used to transfer one or morespecifically desirable traits from one variety, the donor parent, to adeveloped variety called the recurrent parent, which has overall goodcommercial characteristics yet lacks that desirable trait or traits.However, the same procedure can be used to move the progeny toward thegenotype of the recurrent parent, but at the same time retain manycomponents of the nonrecurrent parent by stopping the backcrossing at anearly stage and proceeding with selfing and selection. For example, aZinnia plant may be crossed with another variety to produce afirst-generation progeny plant. The first-generation progeny plant maythen be backcrossed to one of its parent varieties to create a BC₁ orBC₂. Progeny are selfed and selected so that the newly developed varietyhas many of the attributes of the recurrent parent and yet several ofthe desired attributes of the nonrecurrent parent. This approachleverages the value and strengths of the recurrent parent for use in newZinnia varieties.

Therefore, another embodiment is a method of making a backcrossconversion of Zinnia SAKZIN020, comprising the steps of crossing ZinniaSAKZIN020 with a donor plant comprising a desired trait, selecting an F₁progeny plant comprising the desired trait, and backcrossing theselected F₁ progeny plant to Zinnia SAKZIN020. This method may furthercomprise the step of obtaining a molecular marker profile of ZinniaSAKZIN020 and using the molecular marker profile to select for a progenyplant with the desired trait and the molecular marker profile of ZinniaSAKZIN020.

Recurrent Selection and Mass Selection

Recurrent selection is a method used in a plant breeding program toimprove a population of plants. Zinnia SAKZIN020 is suitable for use ina recurrent selection program. The method entails individual plantscross-pollinating with each other to form progeny. The progeny are grownand the superior progeny selected by any number of selection methods,which include individual plant, half-sib progeny, full-sib progeny, andselfed progeny. The selected progeny are cross-pollinated with eachother to form progeny for another population. This population is plantedand again superior plants are selected to cross-pollinate with eachother. Recurrent selection is a cyclical process and therefore can berepeated as many times as desired. The objective of recurrent selectionis to improve the traits of a population. The improved population canthen be used as a source of breeding material to obtain new varietiesfor commercial or breeding use, including the production of a syntheticvariety. A synthetic variety is the resultant progeny formed by theintercrossing of several selected varieties.

Mass selection is a useful technique when used in conjunction withmolecular marker enhanced selection. In mass selection, seeds fromindividuals are selected based on phenotype or selection requiresgrowing a population of plants in a bulk plot, allowing the plants toself-pollinate, harvesting the seed in bulk, and then using a sample ofthe seed harvested in bulk to plant the next generation. Also, insteadof self-pollination, directed pollination could be used as part of thebreeding program.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating plants. A genetically variablepopulation of heterozygous individuals is either identified, or created,by intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Protoplast Fusion

Also known as somatic fusion, this process can be used with SAKZIN020 tocreate hybrids. The resulting hybrid plants have the chromosomes of eachparent and thus the process is useful for incorporating new traits. Theprotoplast fusion technique is well known in the art; see for exampleHamill J. D., Cocking E. C. (1988) Somatic Hybridization of Plants andits Use in Agriculture. In: Pais M. S. S., Mavituna F., Novais J. M.(eds) Plant Cell Biotechnology. NATO ASI Series (Series H: CellBiology), vol 18.

Mutation Breeding

Mutation breeding is another method of introducing new traits intoZinnia SAKZIN020. Mutations that occur spontaneously or are artificiallyinduced can be useful sources of variability for a plant breeder. Thegoal of artificial mutagenesis is to increase the rate of mutation for adesired characteristic. Mutation rates can be increased by manydifferent means including temperature, long-term seed storage, tissueculture conditions, ionizing radiation, such as X-rays, Gamma rays(e.g., cobalt 60 or cesium 137), neutrons, (product of nuclear fissionby uranium 235 in an atomic reactor), Beta radiation (emitted fromradioisotopes such as phosphorus 32 or carbon 14), or ultravioletradiation (preferably from 2500 to 2900 nm); chemical mutagens (such asbase analogues (5-bromo-uracil)), related compounds (8-ethoxy caffeine),antibiotics (streptonigrin), alkylating agents (sulfur mustards,nitrogen mustards, epoxides, ethylenamines, sulfates, sulfonates such asethyl methanesulfonate, sulfones, lactones), sodium azide,hydroxylamine, nitrous acid, methylnitrilsourea, or acridines; TILLING(targeting induced local lesions in genomes), where mutation is inducedby chemical mutagens and mutagenesis is accompanies by the isolation ofchromosomal DNA from every mutated plant line or seed and screening ofthe population of the seed or plants is performed at the DNA level usingadvanced molecular techniques. Once a desired trait is observed throughmutagenesis the trait may then be incorporated into existing germplasmby traditional breeding techniques. Details of mutation breeding can befound in Vainstein, “Breeding for Ornamentals: Classical and MolecularApproaches,” Kluwer Academic Publishers (2002); Sikora, Per, et al.,“Mutagenesis as a Tool in Plant Genetics, Functional Genomics, andBreeding” International Journal of Plant Genomics. 2011 (2011); 13pages. In addition, mutations created in other Zinnia plants may be usedto produce a backcross conversion of Zinnia that comprises suchmutation.

Gene Editing Using CRISPR

Targeted gene editing can be done using CRISPR/Cas9 technology (Saunders& Joung, Nature Biotechnology, 32, 347-355, 2014). CRISPR is a type ofgenome editing system that stands for Clustered Regularly InterspacedShort Palindromic Repeats. This system and CRISPR-associated (Cas) genesenable organisms, such as select bacteria and archaea, to respond to andeliminate invading genetic material. Ishino, Y., et al. J. Bacteriol.169, 5429-5433 (1987). These repeats were known as early as the 1980s inE. coli, but Barrangou and colleagues demonstrated that S. thermophiluscan acquire resistance against a bacteriophage by integrating a fragmentof a genome of an infectious virus into its CRISPR locus. Barrangou, R.,et al. Science 315, 1709-1712 (2007). Many plants have already beenmodified using the CRISPR system. See for example, U.S. ApplicationPublication No. WO2014068346 (György et al., Identification of aXanthomonas euvesicatoria resistance gene from pepper (Capsicum annuum)and method for generating plants with resistance); Martinelli, F. etal., “Proposal of a Genome Editing System for Genetic Resistance toTomato Spotted Wilt Virus” American Journal of Applied Sciences 2014;Noman, A. et al., “CRISPR-Cas9: Tool for Qualitative and QuantitativePlant Genome Editing” Frontiers in Plant Science Vol. 7 Nov. 2016; and“Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis inPetunia” Science Reports Volume 6: February 2016.

Gene editing can also be done using crRNA-guided surveillance systemsfor gene editing. Additional information about crRNA-guided surveillancecomplex systems for gene editing can be found in the followingdocuments, which are incorporated by reference in their entirety: U.S.Application Publication No. 2010/0076057 (Sontheimer et al., Target DNAInterference with crRNA); U.S. Application Publication No. 2014/0179006(Feng, CRISPR-CAS Component Systems, Methods, and Compositions forSequence Manipulation); U.S. Application Publication No. 2014/0294773(Brouns et al., Modified Cascade Ribonucleoproteins and Uses Thereof);Sorek et al., Annu. Rev. Biochem. 82:273-266, 2013; and Wang, S. et al.,Plant Cell Rep (2015) 34: 1473-1476. Therefore, it is another embodimentto use the CRISPR system on Zinnia SAKZIN020 to modify traits andresistances or tolerances to pests, herbicides, diseases, and viruses.

Gene Editing Using TALENs

Transcription activator-like effector nucleases (TALENs) have beensuccessfully used to introduce targeted mutations via repair of doublestranded breaks (DSBs) either through non-homologous end joining (NHEJ),or by homology-directed repair (HDR) and homology-independent repair inthe presence of a donor template. Thus, TALENs are another mechanism fortargeted genome editing using SAKZIN020. The technique is well known inthe art; see for example Malzahn, Aimee et al. “Plant genome editingwith TALEN and CRISPR” Cell & bioscience vol. 7 21. 24 Apr. 2017.

Therefore, it is another embodiment to use the TALENs system on Zinniavariety SAKZIN020 to modify traits and resistances or tolerances topests, herbicides, and viruses.

Other Methods of Genome Editing

In addition to CRISPR and TALENs, two other types of engineerednucleases can be used for genome editing: engineered homingendonucleases/meganucleases (EMNs), and zinc finger nucleases (ZFNs).These methods are well known in the art. See for example, Petilino,Joseph F. “Genome editing in plants via designed zinc finger nucleases”In Vitro Cell Dev Biol Plant. 51(1): pp. 1-8 (2015); and Daboussi,Fayza, et al. “Engineering Meganuclease for Precise Plant GenomeModification” in Advances in New Technology for Targeted Modification ofPlant Genomes. Springer Science+Business. pp 21-38 (2015).

Therefore, it is another embodiment to use engineered nucleases onZinnia variety SAKZIN020 to modify traits and resistances or tolerancesto pests, herbicides, and viruses.

Additional Methods of Transformation

Additional methods include, but are not limited to, expression vectorsintroduced into plant tissues using a direct gene transfer method, suchas microprojectile-mediated delivery, DNA injection, electroporation,and the like. More preferably, expression vectors are introduced intoplant tissues by using either microprojectile-mediated delivery with abiolistic device or by using Agrobacterium-mediated transformation.Transformant plants obtained with the protoplasm of the subject ZinniaSAKZIN020 plants are intended to be within the scope of the embodimentsof the application.

Single-Gene Conversions

When the term Zinnia SAKZIN020 plant is used in the context of anembodiment of the present application, this also includes any singlegene conversions of Zinnia SAKZIN020. The term single gene convertedplant as used herein refers to those Zinnia plants which are developedby a plant breeding technique called backcrossing wherein essentiallyall of the desired morphological and physiological characteristics of avariety are recovered in addition to the single gene transferred intothe variety via the backcrossing technique. Backcrossing methods can beused with one embodiment of the present application to improve orintroduce a characteristic into the variety. The term “backcrossing” asused herein refers to the repeated crossing of a hybrid progeny back tothe recurrent parent, i.e., backcrossing 1, 2, 3, 4, 5, 6, 7, 8, or moretimes to the recurrent parent. The parental Zinnia plant thatcontributes the gene for the desired characteristic is termed thenonrecurrent or donor parent. This terminology refers to the fact thatthe nonrecurrent parent is used one time in the backcross protocol andtherefore does not recur. The parental Zinnia plant to which the gene orgenes from the nonrecurrent parent are transferred is known as therecurrent parent as it is used for several rounds in the backcrossingprotocol (Poehlman & Sleper (1994). In a typical backcross protocol, theoriginal variety of interest (recurrent parent) is crossed to a secondvariety (nonrecurrent parent) that carries the single gene of interestto be transferred. The resulting progeny from this cross are thencrossed again to the recurrent parent and the process is repeated untila Zinnia plant is obtained wherein essentially all of the desiredmorphological and physiological characteristics of the recurrent parentare recovered in the converted plant, in addition to the singletransferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalvariety. To accomplish this, a single gene of the recurrent variety ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphologicalconstitution of the original variety. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross; one ofthe major purposes is to add some commercially important trait or traitsto the plant. The exact backcrossing protocol will depend on thecharacteristic or trait being altered to determine an appropriatetesting protocol. Although backcrossing methods are simplified when thecharacteristic being transferred is a dominant allele, a recessiveallele may also be transferred. In this instance, it may be necessary tointroduce a test of the progeny to determine if the desiredcharacteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new variety but that can beimproved by backcrossing techniques. These traits are well-known in theart.

Introduction of a New Trait or Locus into Zinnia SAKZIN020

Zinnia SAKZIN020 represents a new base of genetics into which a newlocus or trait may be introgressed. Direct transformation andbackcrossing represent two important methods that can be used toaccomplish such an introgression. The term backcross conversion andsingle locus conversion are used interchangeably to designate theproduct of a backcrossing program.

Backcross Conversions of Zinnia SAKZIN020

A backcross conversion of Zinnia SAKZIN020 occurs when DNA sequences areintroduced through backcrossing (Allard, “Principles of Plant Breeding”(1999) with Zinnia SAKZIN020 utilized as the recurrent parent. Bothnaturally occurring and transgenic DNA sequences may be introducedthrough backcrossing techniques. A backcross conversion may produce aplant with a trait or locus conversion in at least two or morebackcrosses, including at least 2 crosses, at least 3 crosses, at least4 crosses, at least 5 crosses, and the like. Molecular marker assistedbreeding or selection may be utilized to reduce the number ofbackcrosses necessary to achieve the backcross conversion. For example,see, Openshaw, S. J., et al., Marker-assisted Selection in BackcrossBreeding, Proceedings Symposium of the Analysis of Molecular Data, CropScience Society of America, Corvallis, Oreg. (August 1994), where it isdemonstrated that a backcross conversion can be made in as few as twobackcrosses.

The complexity of the backcross conversion method depends on the type oftrait being transferred (single genes or closely linked genes ascompared to unlinked genes), the level of expression of the trait, thetype of inheritance (cytoplasmic or nuclear), and the types of parentsincluded in the cross. It is understood by those of ordinary skill inthe art that for single gene traits that are relatively easy toclassify, the backcross method is effective and relatively easy tomanage. See, Allard, “Principles of Plant Breeding” (1999). Desiredtraits that may be transferred through backcross conversion include, butare not limited to, sterility (nuclear and cytoplasmic), fertilityrestoration, drought tolerance, nitrogen utilization, ornamentalfeatures, disease resistance (bacterial, fungal, or viral), insectresistance, and herbicide resistance. In addition, an introgression siteitself, such as an FRT site, Lox site, or other site specificintegration site, may be inserted by backcrossing and utilized fordirect insertion of one or more genes of interest into a specific plantvariety. In some embodiments, the number of loci that may be backcrossedinto Zinnia SAKZIN020 is at least 1, 2, 3, 4, or 5, and/or no more than6, 5, 4, 3, or 2. A single locus may contain several transgenes, such asa transgene for disease resistance that, in the same expression vector,also contains a transgene for herbicide resistance. The gene forherbicide resistance may be used as a selectable marker and/or as aphenotypic trait. A single locus conversion of site-specific integrationsystem allows for the integration of multiple genes at the convertedloci.

The backcross conversion may result from either the transfer of adominant allele or a recessive allele. Selection of progeny containingthe trait of interest is accomplished by direct selection for a traitassociated with a dominant allele. Transgenes or genes transferred viabackcrossing typically function as a dominant single gene trait and arerelatively easy to classify. Selection of progeny for a trait that istransferred via a recessive allele requires growing and selfing thefirst backcross generation to determine which plants carry the recessivealleles. Recessive traits may require additional progeny testing insuccessive backcross generations to determine the presence of the locusof interest. The last backcross generation is usually selfed to givepure breeding progeny for the gene(s) being transferred, although abackcross conversion with a stably introgressed trait may also bemaintained by further backcrossing to the recurrent parent withselection for the converted trait.

In addition, the above process and other similar processes describedherein may be used to produce first generation progeny Zinnia seed byadding a step at the end of the process that comprises crossing ZinniaSAKZIN020 with the introgressed trait or locus with a different plantand harvesting the resultant first generation progeny seed.

Molecular Techniques Using Zinnia SAKZIN020

The advent of new molecular biological techniques has allowed theisolation and characterization of genetic elements with specificfunctions. Traditional plant breeding has principally been the source ofnew germplasm, however, advances in molecular technologies have allowedbreeders to provide varieties with novel and much wanted commercialattributes. Molecular techniques such as transformation are popular inbreeding ornamental plants and well-known in the art. See Vainstein,“Breeding for Ornamentals: Classical and Molecular Approaches,” KluwerAcademic Publishers (2002).

Breeding with Molecular Markers

Molecular markers can also be used during the breeding process for theselection of qualitative traits. For example, markers closely linked toalleles or markers containing sequences within the actual alleles ofinterest can be used to select plants that contain the alleles ofinterest during a backcrossing breeding program. The markers can also beused to select for the genome of the recurrent parent and against thegenome of the donor parent. Using this procedure can minimize the amountof genome from the donor parent that remains in the selected plants. Itcan also be used to reduce the number of crosses back to the recurrentparent needed in a backcrossing program. The use of molecular markers inthe selection process is often called genetic marker enhanced selection.Molecular markers may also be used to identify and exclude certainsources of germplasm as parental varieties or ancestors of a plant byproviding a means of tracking genetic profiles through crosses.Molecular markers, which includes markers identified through the use oftechniques such as Isozyme Electrophoresis, Restriction Fragment LengthPolymorphisms (RFLPs), Randomly Amplified Polymorphic DNAs (RAPDs),Arbitrarily Primed Polymerase Chain Reaction (AP-PCR), DNA AmplificationFingerprinting (DAF), Sequence Characterized Amplified Regions (SCARs),Amplified Fragment Length Polymorphisms (AFLPs), Simple Sequence Repeats(SSRs), and Single Nucleotide Polymorphisms (SNPs), may be used in plantbreeding methods utilizing Zinnia SAKZIN020. See Vainstein, “Breedingfor Ornamentals: Classical and Molecular Approaches,” Kluwer AcademicPublishers (2002).

One use of molecular markers is Quantitative Trait Loci (QTL) mapping.QTL mapping is the use of markers, which are known to be closely linkedto alleles that have measurable effects on a quantitative trait.Selection in the breeding process is based upon the accumulation ofmarkers linked to the positive effecting alleles and/or the eliminationof the markers linked to the negative effecting alleles from the plant'sgenome. See for example, Fletcher, Richard S., et al., “QTL analysis ofroot morphology, flowering time, and yield reveals trade-offs inresponse to drought in Brassica napus” Journal of Experimental Biology.66 (1): 245-256 (2014). QTL markers can also be used during the breedingprocess for the selection of qualitative traits. For example, markersclosely linked to alleles or markers containing sequences within theactual alleles of interest can be used to select plants that contain thealleles of interest during a backcrossing breeding program. The markerscan also be used to select for the genome of the recurrent parent andagainst the genome of the donor parent. Using this procedure canminimize the amount of genome from the donor parent that remains in theselected plants. It can also be used to reduce the number of crossesback to the recurrent parent needed in a backcrossing program. The useof molecular markers in the selection process is often called geneticmarker enhanced selection. Molecular markers may also be used toidentify and exclude certain sources of germplasm as parental varietiesor ancestors of a plant by providing a means of tracking geneticprofiles through crosses.

Production of Double Haploids

The production of double haploids can also be used for the developmentof plants with a homozygous phenotype in the breeding program. Forexample, a Zinnia plant for which Zinnia SAKZIN020 is a parent can beused to produce double haploid plants. Double haploids are produced bythe doubling of a set of chromosomes (1N) from a heterozygous plant toproduce a completely homozygous individual. This can be advantageousbecause the process omits the generations of selfing needed to obtain ahomozygous plant from a heterozygous source. For example, see, Ferrie,Alison M. R., et al., “Review of Doubled Haploidy Methodologies inOrnamental Species” Propagation of Ornamental Plants. 11(2): pp. 63-77(2011).

Thus, an embodiment is a process for making a substantially homozygousZinnia SAKZIN020 progeny plant by producing or obtaining a seed from thecross of Zinnia SAKZIN020 and another Zinnia plant and applying doublehaploid methods to the F₁ seed or F₁ plant or to any successive filialgeneration.

In particular, a process of making seed retaining the molecular markerprofile of Zinnia SAKZIN020 is contemplated, such process comprisingobtaining or producing F₁ seed for which Zinnia SAKZIN020 is a parent,inducing doubled haploids to create progeny without the occurrence ofmeiotic segregation, obtaining the molecular marker profile of ZinniaSAKZIN020, and selecting progeny that retain the molecular markerprofile of Zinnia SAKZIN020.

Expression Vectors for Zinnia Transformation: Marker Genes

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of, or operatively linked to, aregulatory element (for example, a promoter). Expression vectors includeat least one genetic marker operably linked to a regulatory element (forexample, a promoter) that allows transformed cells containing the markerto be either recovered by negative selection, i.e., inhibiting growth ofcells that do not contain the selectable marker gene, or by positiveselection, i.e., screening for the product encoded by the geneticmarker. Many commonly used selectable marker genes for planttransformation are well-known in the transformation arts, and include,for example, genes that code for enzymes that metabolically detoxify aselective chemical agent which may be an antibiotic or an herbicide, orgenes that encode an altered target which is insensitive to theinhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase II (nptII) gene which, when under thecontrol of plant regulatory signals, confers resistance to kanamycin.Another commonly used selectable marker gene is the hygromycinphosphotransferase gene which confers resistance to the antibiotichygromycin.

Selectable marker genes for plant transformation not of bacterial origininclude, for example, mouse dihydrofolate reductase, plant5-enolpyruvylshikimate-3-phosphate synthase, and plant acetolactatesynthase (Eichholtz, et al., Somatic Cell Mol. Genet., 13:67 (1987);Shah, et al., Science, 233:478 (1986); Charest, et al., Plant Cell Rep.,8:643 (1990)).

Another class of marker genes for plant transformation requiresscreening of presumptively transformed plant cells, rather than directgenetic selection of transformed cells, for resistance to a toxicsubstance such as an antibiotic. These genes are particularly useful toquantify or visualize the spatial pattern of expression of a gene inspecific tissues and are frequently referred to as reporter genesbecause they can be fused to a gene or gene regulatory sequence for theinvestigation of gene expression. Commonly used marker genes forscreening presumptively transformed cells include β-glucuronidase (GUS),β-galactosidase, luciferase, and chloramphenicol acetyltransferase(Jefferson, R. A., Plant Mol. Biol. Rep., 5:387 (1987); Teeri, et al.,EMBO J., 8:343 (1989); Koncz, et al., Proc. Natl. Acad. Sci. USA, 84:131(1987); DeBlock, et al., EMBO J., 3:1681 (1984)).

Expression Vectors for Zinnia Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotidesequence comprising a regulatory element (for example, a promoter).Several types of promoters are well known in the transformation arts asare other regulatory elements that can be used alone or in combinationwith promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred.”Promoters that initiate transcription only in a certain tissue arereferred to as “tissue-specific.” A “cell-type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may affect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell-type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter that is active under mostenvironmental conditions. Many types of promoters are well known in theart.

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of a protein produced by transgenes to a subcellularcompartment, such as the chloroplast, vacuole, peroxisome, glyoxysome,cell wall, or mitochondrion, or for secretion into the apoplast, isaccomplished by means of operably linking the nucleotide sequenceencoding a signal sequence to the 5′ and/or 3′ region of a gene encodingthe protein of interest. Targeting sequences at the 5′ and/or 3′ end ofthe structural gene may determine during protein synthesis andprocessing where the encoded protein is ultimately compartmentalized.Many signal sequences are well-known in the art. See, for example,Becker, et al., Plant Mol. Biol., 20:49 (1992); Knox, C., et al., PlantMol. Biol., 9:3-17 (1987); Lerner, et al., Plant Physiol., 91:124-129(1989); Frontes, et al., Plant Cell, 3:483-496 (1991); Matsuoka, et al.,Proc. Natl. Acad. Sci., 88:834 (1991); Gould, et al., J Cell. Biol.,108:1657 (1989); Creissen, et al., Plant J., 2:129 (1991); Kalderon, etal., Cell, 39:499-509 (1984); Steifel, et al., Plant Cell, 2:785-793(1990).

Foreign Protein Genes: Transformation

Various promoters, targeting sequences, enhancing sequences, and otherDNA sequences can be inserted into the genome for the purpose ofaltering the expression of genes

Gene Silencing and Altering Gene Expression

Many techniques for altering gene expression are well-known to one ofskill in the art, including, but not limited to, knock-outs (such as byinsertion of a transposable element such as Mu (Vicki Chandler, TheMaize Handbook, Ch. 118 (Springer-Verlag 1994)) or other geneticelements such as a FRT, Lox, or other site specific integration sites;antisense technology (see, e.g., Sheehy, et al., PNAS USA, 85:8805-8809(1988) and U.S. Pat. Nos. 5,107,065, 5,453,566, and 5,759,829);co-suppression (e.g., Taylor, Plant Cell, 9:1245 (1997); Jorgensen,Trends Biotech., 8(12):340-344 (1990); Flavell, PNAS USA, 91:3490-3496(1994); Finnegan, et al., Bio/Technology, 12:883-888 (1994); Neuhuber,et al., Mol. Gen. Genet., 244:230-241 (1994)); RNA interference (Napoli,et al., Plant Cell, 2:279-289 (1990); U.S. Pat. No. 5,034,323; Sharp,Genes Dev., 13:139-141 (1999); Zamore, et al., Cell, 101:25-33 (2000);Montgomery, et al., PNAS USA, 95:15502-15507 (1998)), virus-induced genesilencing (Burton, et al., Plant Cell, 12:691-705 (2000); Baulcombe,Curr. Op. Plant Bio., 2:109-113 (1999)); target-RNA-specific ribozymes(Haseloff, et al., Nature, 334:585-591 (1988)); hairpin structures(Smith, et al., Nature, 407:319-320 (2000); U.S. Pat. Nos. 6,423,885,7,138,565, 6,753,139, and 7,713,715); MicroRNA (Aukerman & Sakai, PlantCell, 15:2730-2741 (2003)); ribozymes (Steinecke, et al., EMBO J.,11:1525 (1992); Perriman, et al., Antisense Res. Dev., 3:253 (1993));oligonucleotide mediated targeted modification (e.g., U.S. Pat. Nos.6,528,700 and 6,911,575); Zn-finger targeted molecules (e.g., U.S. Pat.Nos. 7,151,201, 6,453,242, 6,785,613, 7,177,766 and 7,788,044);transposable elements (e.g. Dubin, M. J., et al., Transposons: ablessing curse, Current opinion in plant biology, Vol: 42, Page: 23-29,2018 and Eric T. Johnson, Jesse B. Owens & Stefan Moisyadi (2016) Vastpotential for using the piggyBac transposon to engineer transgenicplants at specific genomic locations, Bioengineered, 7:1, 3-6); andother methods or combinations of the above methods known to those ofskill in the art.

Additional Transformation Embodiments

The foregoing methods for transformation may be used for producing atransgenic variety. The transgenic variety could then be crossed withanother (non-transformed or transformed) variety in order to produce anew transgenic variety. Alternatively, a genetic trait that has beenengineered into a particular Zinnia line using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite variety into an elitevariety, or from a variety containing a foreign gene in its genome intoa variety or varieties that do not contain that gene. As used herein,“crossing” can refer to a simple x by y cross or the process ofbackcrossing depending on the context.

Likewise, by means of one embodiment, commercially important genes canbe expressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of commercialinterest, including, but not limited to, genes that confer resistance topests or disease, genes that confer resistance to an herbicide, genesthat confer or contribute to a value-added or desired trait, genes thatcontrol male sterility, genes that create a site for site specific DNAintegration, and genes that affect abiotic stress resistance. Manyhundreds if not thousands of different genes are known and couldpotentially be introduced into a Zinnia plant according to theinvention. Non-limiting examples of particular genes and correspondingphenotypes one may choose to introduce into a Zinnia plant include oneor more genes for insect tolerance, such as a Bacillus thuringiensis(Bt.) gene, pest tolerance such as genes for fungal disease control,herbicide tolerance such as genes conferring glyphosate tolerance, andgenes for quality improvements such as environmental or stresstolerances, or any desirable changes in plant physiology, growth,development, morphology or plant product(s). For example, structuralgenes would include any gene that confers insect tolerance including butnot limited to a Bacillus insect control protein gene as described in WO99/31248, herein incorporated by reference in its entirety, U.S. Pat.No. 5,689,052, herein incorporated by reference in its entirety, U.S.Pat. Nos. 5,500,365 and 5,880,275, herein incorporated by reference intheir entirety. In another embodiment, the structural gene can confertolerance to the herbicide glyphosate as conferred by genes including,but not limited to Agrobacterium strain CP4 glyphosate resistant EPSPSgene (aroA:CP4) as described in U.S. Pat. No. 5,633,435, hereinincorporated by reference in its entirety, or glyphosate oxidoreductasegene (GOX) as described in U.S. Pat. No. 5,463,175, herein incorporatedby reference in its entirety. Alternatively, the DNA coding sequencescan affect these phenotypes by encoding a non-translatable RNA moleculethat causes the targeted inhibition of expression of an endogenous gene,for example via antisense- or cosuppression-mediated mechanisms (see,for example, Bird et al., Biotech. Gen. Engin. Rev., 9:207, 1991). TheRNA could also be a catalytic RNA molecule (i.e., a ribozyme) engineeredto cleave a desired endogenous mRNA product (see for example, Gibson andShillito, Mol. Biotech., 7:125, 1997). Thus, any gene which produces aprotein or mRNA which expresses a phenotype or morphology change ofinterest is useful for the practice of one or more embodiments.

Tissue Culture

Further reproduction of the variety can occur by tissue culture andregeneration. Tissue culture of various tissues of ornamental plants andZinnia SAKZIN020 and regeneration of plants therefrom is well-known andwidely published. For example, reference may be had to H. Mahmoodzadeh,F. Abbasi and S. Rohani, 2010. In vitro Germination and Early SeedlingGrowth of Zinnia elegans. Research Journal of Environmental Sciences, 4:407-413; Valla Rego, Luciana et al., Crop Breeding and AppliedTechnology. 1(3): 283-300 (2001); Komatsuda, T., et al., Crop Sci.,31:333-337 (1991); Stephens, P. A., et al., Theor. Appl. Genet.,82:633-635 (1991); Komatsuda, T., et al., Plant Cell, Tissue and OrganCulture, 28:103-113 (1992); Dhir, S., et al., Plant Cell Reports,11:285-289 (1992); Pandey, P., et al., Japan J Breed., 42:1-5 (1992);and Shetty, K., et al., Plant Science, 81:245-251 (1992). Thus, anotherembodiment is to provide cells which upon growth and differentiationproduce Zinnia plants having the physiological and morphologicalcharacteristics of Zinnia SAKZIN020 described in the presentapplication.

Regeneration refers to the development of a plant from tissue culture.The term “tissue culture” indicates a composition comprising isolatedcells of the same or a different type or a collection of such cellsorganized into parts of a plant. Exemplary types of tissue cultures areprotoplasts, calli, plant clumps, and plant cells that can generatetissue culture that are intact in plants or parts of plants, such aspollen, ovules, embryos, protoplasts, meristematic cells, callus,pollen, leaves, ovules, anthers, cotyledons, hypocotyl, pistils, roots,root tips, flowers, seeds, petiole, shoot, or stems, and the like. Meansfor preparing and maintaining plant tissue culture are well-known in theart.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize certain modifications,permutations, additions and sub-combinations thereof. It is thereforeintended that the following appended claims and claims hereafterintroduced are interpreted to include all such modifications,permutations, additions, and sub-combinations as are within their truespirit and scope.

One or more aspects may be embodied in other specific forms withoutdeparting from its spirit or essential characteristics. The describedembodiments are to be considered in all respects only as illustrativeand not restrictive. The scope of the embodiments is, therefore,indicated by the appended claims rather than by the foregoingdescription. All changes which come within the meaning and range ofequivalency of the claims are to be embraced within their scope. Theforegoing discussion of the embodiments has been presented for purposesof illustration and description. The foregoing is not intended to limitthe embodiments to the form or forms disclosed herein. In the foregoingDetailed Description for example, various features of the embodimentsare grouped together in one or more embodiments for the purpose ofstreamlining the disclosure. This method of disclosure is not to beinterpreted as reflecting an intention that the claimed embodimentsrequire more features than are expressly recited in each claim. Rather,as the following claims reflect, inventive aspects lie in less than allfeatures of a single foregoing disclosed embodiment. Thus, the followingclaims are hereby incorporated into this Detailed Description, with eachclaim standing on its own as a separate preferred embodiment.

Moreover, though the description of the embodiments has includeddescription of one or more embodiments and certain variations andmodifications, other variations and modifications are within the scopeof the embodiments (e.g., as may be within the skill and knowledge ofthose in the art, after understanding the present disclosure). It isintended to obtain rights which include alternative embodiments to theextent permitted, including alternate, interchangeable and/or equivalentstructures, functions, ranges or acts to those claimed, whether or notsuch alternate, interchangeable and/or equivalent structures, functions,ranges or acts are disclosed herein, and without intending to publiclydedicate any patentable subject matter.

The use of the terms “a,” “an,” and “the,” and similar referents in thecontext of describing the embodiments (especially in the context of thefollowing claims) are to be construed to cover both the singular and theplural, unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. Forexample, if the range 10-15 is disclosed, then 11, 12, 13, and 14 arealso disclosed. All methods described herein can be performed in anysuitable order unless otherwise indicated herein or otherwise clearlycontradicted by context. The use of any and all examples, or exemplarylanguage (e.g., “such as”) provided herein, is intended merely to betterilluminate the embodiments and does not pose a limitation on the scopeof the embodiments unless otherwise claimed. No language in thespecification should be construed as indicating any non-claimed elementas essential to the practice one or more embodiments.

Deposit Information

A deposit of the Sakata Seed Corporation proprietary Zinnia lineSAKZIN020 seed disclosed above and recited in the appended claims hasbeen made with and accepted under the Budapest Treaty by the NationalCollections of Industrial, Food and Marine Bacteria (NCIMB), 23 StMachar Drive, Aberdeen, Scotland, AB24 3RY, United Kingdom. The date ofdeposit was Feb. 19, 2020. The NCIMB No. is 43585. The deposit of seedwas taken from the same deposit maintained by Sakata Seed Corporationsince prior to the filing date of this application. The deposit will bemaintained in the NCIMB depository for a period of 30 years, or 5 yearsafter the most recent request, or for the enforceable life of thepatent, whichever is longer, and will be replaced if necessary, duringthat period. Upon issuance, all restrictions on the availability to thepublic of the deposit will be irrevocably removed consistent with all ofthe requirements of 37 C.F.R. §§ 1.801-1.809.

What is claimed is:
 1. A seed of Zinnia line SAKZIN020, wherein arepresentative sample of seed of said Zinnia line was deposited underNCIMB No.
 43585. 2. A plant, or a plant part thereof produced by growingthe seed of claim 1, wherein the plant or plant part comprises at leastone cell of Zinnia line SAKZIN020.
 3. A Zinnia plant, or a part thereof,having all of the physiological and morphological characteristics of theplant of claim
 2. 4. A tissue or cell culture of regenerable cellsproduced from the plant of claim
 1. 5. The tissue or cell culture ofclaim 4, comprising tissues or cells from a plant part selected from thegroup consisting of leaves, pollen, embryos, cotyledons, hypocotyl,meristematic cells, roots, root tips, pistils, anthers, flowers, andstems.
 6. A Zinnia plant regenerated from the tissue or cell culture ofclaim 5, wherein said plant has all of the morphological andphysiological characteristics of Zinnia SAKZIN020.
 7. A method ofproducing a hybrid Zinnia seed, wherein the method comprises crossing aplant of Zinnia SAKZIN020, a representative sample of seed of saidZinnia line was deposited under NCIMB No. 43585, with another SAKZIN020plant or a different zinnia plant and harvesting the resultant Zinniaseed.
 8. An F₁ Zinnia seed produced by the method of claim
 7. 9. An F₁Zinnia plant, or a part thereof, produced by growing the seed of claim8, wherein said part is leaves, pollen, embryos, cotyledons, hypocotyl,meristematic cells, roots, root tips, pistils, anthers, flowers, andstems.
 10. A plant of Zinnia line SAKZIN020, wherein a representativesample of seed of said Zinnia line was deposited under NCIMB No. 43585.11. A method of vegetatively propagating the plant of claim 10,comprising the steps of: collecting tissue or cells capable of beingpropagated from said plant; cultivating said tissue or cells to obtainproliferated shoots; and rooting said proliferated shoots to obtainrooted plantlets; or cultivating said tissue or cells to obtainproliferated shoots, or to obtain plantlets.
 12. A Zinnia plant producedby growing the plantlets or proliferated shoots of claim
 11. 13. Amethod for producing an embryo or seed, wherein the method comprisescrossing the plant of claim 10 with another plant and harvesting theresultant embryo or seed.
 14. A method of determining the genotype ofthe Zinnia plant of claim 10, wherein said method comprises obtaining asample of nucleic acids from said plant and detecting in said nucleicacids a plurality of polymorphisms.
 15. A method of producing a Zinniaplant resistant to the group consisting of herbicides, insecticides, anddisease, wherein the method comprises transforming the Zinnia plant ofclaim 10 with a transgene, and wherein said transgene confers resistanceto an herbicide, insecticide, or disease.
 16. An herbicide, insecticide,or disease resistant plant produced by the method of claim
 15. 17. Amethod for developing a Zinnia plant in a plant breeding program,comprising applying plant breeding techniques comprising crossing,recurrent selection, mutation breeding, wherein said mutation breedingselects for a mutation that is spontaneously or naturally induced orartificially induced, backcrossing, pedigree breeding, marker enhancedselection, haploid/double haploid production, or transformation to theZinnia plant of claim 10, or its parts, wherein application of saidtechniques results in development of a Zinnia plant.
 18. A method ofintroducing a mutation into the genome of Zinnia plant SAKZIN020, saidmethod comprising mutagenesis of the plant, or plant part thereof, ofclaim 10, wherein said mutagenesis is selected from the group consistingof temperature, long-term seed storage, tissue culture conditions,ionizing radiation, chemical mutagens, or targeting induced locallesions in genomes, and wherein the resulting plant comprises at leastone genome mutation.
 19. A method of editing the genome of Zinnia plantSAKZIN020, said method comprising mutagenesis of the plant, or plantpart thereof, of claim 10, wherein said method is selected from thegroup comprising zinc finger nucleases, transcription activator-likeeffector nucleases (TALENs), engineered homingendonucleases/meganucleases, and the clustered regularly interspacedshort palindromic repeat (CRISPR)-associated protein9 (Cas9) system. 20.A Zinnia plant produced by the method of claim
 19. 21. A Zinnia seedproduced by growing the plant of claim
 10. 22. A method of producing aZinnia plant, or part thereof, produced by growing the seed of claim 21.23. A method for producing an embryo or seed, wherein the methodcomprises crossing the plant of claim 10 with another plant andharvesting the resultant embryo or seed.
 24. The method of claim 23,further comprising producing a plant, or a part thereof, from theresultant embryo or seed.